|
Fig. 1
shha expression is controlled by cyp26a1-expressing epidermal niches. Lateral expansion of the cyp26a1-expressing niches and misguidance of preosteoblasts precede fusion of sister rays. (A) Overview of a regenerating fin. (B) Schematized representation of the amputation levels. (C-E,L) Amputations in proximity to bifurcations cause fusion of cyp26a1 expression domains (arrowhead in L), followed by spreading of preosteoblasts into the interray separating the two sister rays (arrowhead in E) and fusion of sister rays (arrow in C). (C) Alizarin Red staining at 9 dpa. Arrowheads indicate branching sites. (D) Distance range between sister rays for separated and fused rays. (E) IHC for ZNS-5 and GFP in osc:gfp fish at 2.5 dpa. (F,G) WISH and ISH on sections for cyp26a1 reveals expression in the proximal basal epidermal layer (arrowhead) adjacent to preosteoblasts (asterisks) at 2 and 3 dpa. (G) aldh1a2 is expressed in fibroblast-derived blastema cells, but not in preosteoblasts adjacent to cyp26a1-expressing cells. (H,I) RA and R115866 injection downregulates shha and ptch2. Transcript levels 6 or 8 hours after injection at 4 dpa measured by qPCR (H); WISH for shha 12 hours after R115866 injection at 4 dpa (I). (J,K) Overexpression of cyp26a1 in hsp70I:cyp26a1 fish at 4 dpa causes upregulation of shha expression and lateral expansion of the expression domains. Arrows mark the lateral expression limits. Of note, shha in wild-type controls is already expressed in subdomains due to the upcoming bifurcation event. (L) WISH for cyp26a1 at 1.5 dpa. Double-headed arrows in E and L: distance between sister rays. Asterisk marks the gap between the two subdomains. Data are represented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001. Dashed lines indicate amputation plane. Scale bars: 100µm. h, hours; hs, heat shock.